Contributions to the rotifer Fauna of West Bengal. Part I. Family Lecanidae

In the present study thirty six lecanid rotifers, spreading over thirty species, have been recorded and illustrated. A new combination i.e., Lecane luna f. dorsicalis has been proposed. The validity of the number of toes as character of generic importance has been examined.     

Competitive interaction between two functional S-haplotypes confer self-compatibility on tetraploid Chinese cherry (Prunus pseudocerasus Lindl. CV. Nanjing Chuisi)

Self-incompatibility (SI) has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility (GSI). In the present study, four PpsS-haplotypes (Prunus pseudocerasus S-haplotypes) comprising at least two genes, i.e., PpsS-RNase (P. pseudocerasus S-RNase) and PpsSFB (P. pseudocerasus S-haplotype-specific F-box) have been successfully isolated in tetraploid P. pseudocerasus Lindl. CV. Nanjing Chuisi ("NC") which exhibited self-compatibility (SC), and its S-genotype was determined as S-1/S-3'/S-5/S-7. These PpsS-RNases, which were expressed exclusively in style, shared the typical structural features with S-RNases from other Prunus species exhibiting GSI. All PpsSFBs showed similar structure characteristics of SFBs from other Prunus species, and matched with the necessary conditions for pollen S-determinant. No mutations leading to dysfunction of S-haplotype were found in their full-length c-DNA sequences, except for PpsS-3'-haplotype which was not amplified by PCR. These four S-haplotypes complied with tetrasomic inheritance. Diploid pollen grains with S-genotypes S-7/S-1, S-7/S-5 and S-1/S-5 can grow the full length of the style after self-pollination, while pollen grains with S-3'/S-7, S-3'/S-1 and S-3'/S-5 cannot. These results suggest that PpsS-haplotypes-1, -5 and -7 are functional, and that competitive interaction between two of them confer self-compatibility on cultivar "NC". Furthermore, in terms of recognition specificity, diploid pollen grains carrying PpsS-3'-haplotype are equal to monoploid pollen grains carrying the other functional S-haplotype.     

Effects of experimental infection with Eimeria bovis on the balance of sodium, potassium and water in calves

Balance trials were performed to investigate the effects of experimental Eimeria bovis coccidiosis on the metabolism of water, sodium and potassium in calves. Non-infected pair-fed controls and controls fed according to plan were included in the study to allow differentiation between the effects due to infection and due to changes in feed intake. Primary infection with 5 × 10⁴ (group A) or 1 × 10⁵ (group B) oocysts caused mild diarrhoea in three out of four group A calves and mild to severe haemorrhagic diarrhoea in all five group B calves. Losses of sodium and potassium via faeces tended to increase in the infected calves during patency and apparent digestibility (AD) of these minerals was comparably low. In the urine of the infected calves the Na/K-ratio decreased due to a reduced urinary excretion of sodium. The retention (RT) of sodium was particularly high in the calves that had received the higher oocyst dose. Potassium RT did not underlie significant changes during the course of coccidiosis. In the infected calves the plasma level of sodium was reduced transiently while the level of potassium remained fairly stable. Infections with the higher oocyst dose caused a distinct reduction of fluid excretion via urine which compensated for the increased faecal water losses during severe diarrhoea. Reinfection of the group A calves with 1 × 10⁵ oocysts did not cause any significant metabolic impairment. The results of this study indicate that although acute sublethal bovine coccidiosis alters electrolyte and water metabolism the overall balance of electrolytes and water is largely maintained by physiologic adaptation.     

High-and low-intensity photosystems in Phycomyces phototropism: Effects of mutations in genes madA,madB, and madC

Sporangiophores of Phycomyces blakesleeanus Burgeff that have been grown in darkness and are then suddenly exposed to unilateral light show a two-step bending response rather than a smooth, monotonic response found in light-adapted specimens (Galland and Lipson, 1987, Proc. Natl. Acad. Sci. USA 84, 104–108). The stepwise bending is controlled by two photosystems optimized for the low-and high-intensity ranges. These two photosystems have now been studied in phototropism mutants with defects in genes madA, madB, and madC. All three mutations raise the threshold of the low-intensity (low-fluence) photosystem by about 10⁶-fold and that of the high-intensity (high-fluence) system by about 10³-fold. Estimates for the light-adaptation time constants of the low-and high-intensity photosystems show that the mutants are affected in adaptation. In the mutants, the light-adaptation kinetics are only slightly affected in the low-intensity photosystem but, for the high-intensity photosystem, the kinetics are considerably slower than in the wild type.Abbreviations WT wild type     

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Sporangia were accumulated in autotrophically and mixotrophically growing cultures of the Chlamydomonas reinhardtii mutant strain ls entering the stationary phase. Such an accumulation of sporangia was never observed in stationary-phase cultures of wildtype strains. Sporangia harvested from stationary-phase cultures of the mutant strain ls released their zoospores after being resuspended in fresh culture medium. Liberation of zoospores was also observed during fixation of these sporangia with glutaraldehyde and OsO₄. Release of zoospores during fixation was prevented by pretreatment with 3 mol·l^–1 LiCl. Ultrastructural analyses of these LiCl-pretreated sporangia revealed that they contained abnormal sporangial walls: sporangia containing sporangia and sporangia surrounded by additional multilayered cell walls have been observed. Similar abnormal cell-wall structures were found in sporangia accumulated at the end of the dark period, when the mutant strain ls was grown photoautotrophically under a 12 h light-12 h dark regime with suboptimal aeration. When grown under optimal conditions, this particular mutant did not show any abnormal wall structures.This work has been supported by a grant from the Deutsche Forschungsgemeinschaft. The authors thank Mrs. C. Adami for the photographic work.     

Influence of the<Emphasis Type="Italic">SMT2</Emphasis> knock-out on hypocotyl elongation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inhibitors are very important in the study of hormone function. Brasinazole (Brz) is a specific inhibitor of brassinosteroids (BRs) biosynthesis. To expand our knowledge of the molecular mechanisms of plant steroid signaling, we performed genetic screening using medium containing Brz under dark conditions. Mutants insensitive to Brz developlonger hypocotyls than their wild type counterparts. We isolatedabz453 as a Brz insensitive mutant. TAIL-PCR and the segregation ratio of T2 plants indicated a single T-DNA insertion at the 24-Sterol C-methyltransferase (SMT2) gene in theabz453 mutant. Recapitulation for putative FCP serine phosphatase (FSP), the gene neighboringSMT2, indicated no significant phenotypes, but theSMT2 anti-sense (SMT2-AS) line developed longer hypocotyls than the wild type in medium containing Brz. Additionally, theSMT2-AS line displayed similar phenotypes to theabz453 line in soil including enhanced growth and smaller silique. Theabz453 andSMT2-AS mutants showed phenotypes similar to those of wild type in medium containing benzylaminopurine, pacrobutrazol and ACC (precursor for ethylene) under dark conditions. However, when brassinolide (BL) dose response was observed, theabz453 andSMT2-AS lines showed higher sensitivity than wild type. Theabz453/det2 andabz453/bri1-119 double mutants showed enhanced growth compared to thedet2 andbri1-119 line under both dark and light conditions. Specially, in dark conditions double mutants displayed nearly 2- and 1.5-fold longer hypocotyls thandet2 andbri1-119 plants. Brz insensitivity to theSMT2 knock-out mutant and phenotypes of double mutants indicate that not only do BRI1 and DET2 influence the BRs response, as evidenced by hypocotyl elongation, but another sterol derived signals may also be affected in mutants, suggesting that another pathway is involved in hypocotyl elongation due to SMT2.     

The wings ofBombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm,Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development inB. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike inDrosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adultDrosophila wings. Partially excised wing discs did not showin situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early inB. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades inB. mori unlike evagination of only the pouch region as wing blades seen inDrosophila.     

A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers

Conservation of Lethal-leaf spot 1 (Lls1) lesion mimic gene in land plants including moss is consistent with its recently reported function as pheophorbide a oxygenase (Pao) which catalyzes a key step in chlorophyll degradation (Pruzinska et al., 2003). A bioinformatics survey of complete plant genomes reveals that LLS1(PAO) belongs to a small 5-member family of non-heme oxygenases defined by the presence of Rieske and mononuclear iron-binding domains. This gene family includes chlorophyll a oxygenase (Cao), choline monooxygenase (Cmo), the gene for a 55 kDa protein associated with protein transport through the inner chloroplast membrane (Tic 55) and a novel 52 kDa protein isolated from chloroplasts (Ptc 52). Analysis of gene structure reveals that these genes diverged prior to monocot/dicot divergence. Homologues of LLS1(PAO), CAO, TIC55 and PTC52 but not CMO are found in the genomes of several cyanobacteria. LLS1(PAO), PTC52, TIC55 and a set of related cyanobacterial homologues share an extended carboxyl terminus containing a novel F/Y/W-x(2)-H-x(3)-C-x(2)-C motif not present in CAO. These proteins appear to have evolved during the transition to oxygenic photosynthesis to play various roles in chlorophyll metabolism. In contrast, CMO homologues are found only in plants and are most closely related to aromatic ring-hydroxylating enzymes from soil-dwelling bacteria, suggesting a more recent evolution of this enzyme, possibly by horizontal gene transfer. Our phylogenetic analysis of 95 extant non-heme dioxygenases provides a useful framework for the classification of LLS1(PAO)-related non-heme oxygenases.     

In vitro production of pectolytic and cellulolytic enzymes byColletotrichum lindemuthianum isolated from soybean grown in Saudi Arabia

Colletotrichum lindemuthianum isolated from soybean in Saudi Arabia produced polygalacturonase, pectin methylesterase, pectin trans-eliminase and carboxymethylcellulasein vitro. Polygalacturonase showed maximaum activity at 30 to 35°C and pH 4.0 to 5.0. The absorption maximum for pectin trans-eliminase reaction products was at approximately 548 nm. The polygalacturonase and pectin trans-eliminase activities increased with culture age. The degradation of carboxymethylcellulose was also demonstrated.     

Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-biosynthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Medaka double mutants for color interfere and leucophore free: characterization of the xanthophore–somatolactin relationship using the leucophore free gene

Somatolactin (SL) plays an essential role in body-color regulation in medaka and is encoded by the color interfere (ci) locus. The ci mutant fish possess constitutively increased numbers of leucophores and a concomitant decrease in visible xanthophores. However, the mechanism of action of SL on these cell types, and the role of SL in body-color regulation in other species, is unknown. In this study, we verified an SL–xanthophore relationship in ci mutant fish using the leucophore free (lf) gene. Histological observation of lf larvae indicated that these mutants do not possess differentiated leucophores. The ci–lf double mutant, whose genotype was confirmed using DNA markers, lacked leucophores; however, the number of xanthophores remained low, demonstrating that leucophores are not necessary for mediating SL signaling to xanthophores. This finding suggests a conserved function for SL in xanthophore regulation across species, rather than the evolution of a medaka-specific and leucophore-dependent role of SL in body-color regulation. Our results also demonstrate that the lf gene has an indispensable role in leucophore development epistatic to SL signaling. The lf gene has not been cloned. The high-resolution recombination map surrounding the lf locus constructed in this study, together with medaka whole genome sequences that will be released soon, will allow the rapid cloning of the lf gene by forward genetic approaches.     

Spore production and mycorrhizal development in various tropical crop hosts infected with Glomus clarum

Plant growth, mycorrhizal development and vesicular arbuscular spore production were examined in five tropical crop host species inoculated with Glomus clarum and grown in a glasshouse. In one of the two experiments, sequential harvests of maize, sorghum and chickpea were made in order to study spore production in relation to plant growth and mycorrhizal development. Spore numbers in each of these hosts increased at a fairly constant rate until maximum plant dry weight, when spore production ceased. Sorghum and maize produced considerably more spores than chickpea, with spore numbers being closely correlated with mycorrhizal root length. In the second experiment, Glomus clarum was cultured on each of maize, millet, sorghum, groundnut and chickpea for three consecutive generations before cross-inoculation of the spores from each host onto all five hosts. Sporulation with respect to host size was generally greatest when the inoculum used to infect a host had been produced on that host. The growth-promoting effects of the fungus were not influenced by the source of the inoculum. More spores were produced on the cereals than the legumes. Differences in spore numbers amongst hosts and plant generations were apparently influenced mainly by infected root length and by the growth period.     

Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog

With more than 50 genes in human, keratins make up a large gene family, but the evolutionary pressure leading to their diversity remains largely unclear. Nevertheless, this diversity offers a means to examine the evolutionary relationships among organisms that express keratins. Here, we report the analysis of keratins expressed in two cyprinid fishes, goldfish and carp, by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot binding assay, and immunoblotting. We further explore the expression of keratins by immunofluorescence microscopy. Comparison is made with the keratin expression and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of molecular weights and isoelectric points, with a similar overall pattern on two-dimensional gels. In addition, immunofluorescence microscopy studies of goldfish and carp tissues have revealed the expression of keratins in both epithelial and mesenchymally derived tissues, as reported previously for zebrafish and trout. We conclude that keratin expression is qualitatively similar among these fishes, with goldfish and carp patterns being more similar to each other than to zebrafish, and the cyprinid fishes being more similar to each other than to the salmonid trout. Because of the detected similarity of keratin expression among the cyprinid fishes, we propose that, for certain experiments, they are interchangeable. Although the zebrafish distinguishes itself as being a developmental and genetic/genomic model organism, we have found that the goldfish, in particular, is a more suitable model for both biochemical and histological studies of the cytoskeleton, especially since goldfish cytoskeletal preparations seem to be more resistant to degradation than those from carp or zebrafish. This work was supported by grants to J.M. from the Stiftung Rheinland Pfalz für Innovation (836-386261/138) and the Deutsche Forschungsgemeinschaft (Ma 843/5-1) and a grant to D.G. from the National Science Foundation (INT-0078261).     

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae)

Scenedesmus obliquus, strain 633, which synthesizes ketocarotenoids and sporopollenin, also forms pink-red-colored cell walls. Both the cell walls left over after autospore liberation and those from homogenates of disrupted green cells have similar carotenoid pigmentation. Canthaxanthin, astaxanthin, an unidentified ketocarotenoid, and lutein were found as integral cell wall components. They are bound to the outer (trilaminar) layer of the complete cell wall which also contains sporopollenin.Abbreviations CWH complete cell walls isolated from the homogenates - CWM maternal cell walls accumulated in the medium - KC ketocarotenoid - SC secondary carotenoids - SP sporopollenin     

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30muM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10muM) and Pseudomonas aeruginosa (MIC=31muM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC(50) approximately 20muM) against the promastigote and amastigote stages of Leishmania infantum.     

The accumulation and assimilation of dimethylselenide by four plant species

Plants of Agrostis tenuis Sibth., Hordeum vulgare L., Lycopersicon esculentum Mill. and Raphanus sativus L. were grown hydroponically in sealed systems and fumigated with 8 g m^-3 [⁷⁵Se]-dimethylselenide. The accumulation of ⁷⁵Se was measured and the shoot tissues were extracted to examine the products of the ⁷⁵Se assimilation. Characteristic differences were observed between species in the accumulation of ⁷⁵Se and the transport from shoots to roots. High-voltage electrophoresis and chromatography of extracts made with 80% aqueous ethanol revealed the presence of inorganic selenite as an assimilation product as well as the selenium analogues of glutathione and methionine. Extensive incorporation of ⁷⁵Se into protein-bound selenomethionine was observed in all plant species.Abbreviation DMSe dimethylselenide     

Maltose excretion by the symbiotic Chlorella of the heliozoan Acanthocystis turfacea

Chlorella sp. strain 3.83, a symbiotic Chlorella isolated from the heliozoan Acanthocystis turfacea, excreted between 8% and 16% of assimilated ¹⁴CO₂ as maltose in the light (15000 lx), with a pH optimum around 4.8. This percentage increased when the illuminance was lowered (36% at 1700 lx). Release of [¹⁴C]maltose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [¹⁴C]maltose (5 mM) was taken up by the cells with a rate <2% of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.Abbreviations DES diethylstilbestrol - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - p.c. packed cells This work was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Doris Meindl for skillful experimental help.     

The Synthesis and Cytotoxic Activity of D-Ribofuranosides and 2-Deoxy-D-Ribofuranosides of Substituted Bis(indolyl)furan, Bis(indolyl)pyrrole, and Indolo[2,3-a]carbazole Derivatives

1-(2,3,5-Tri-O-acetyl)--D-ribofuranosyl indole, the key compound in the synthesis of glycosides with the bis(indole) aglycone, was obtained for the first time by the indoline–indole method. There were synthesized 3-(1-methylindol-3-yl)-4-(1-glycosylindol-3-yl)furan(or pyrrole)-2,5-diones containing the residue of -D-ribofuranose or 2-deoxy--D-ribofuranose and analogous glycosides of indolo[2,3-a]furano(or pyrrolo)[3,4-]carbazol-5,7-diones, which are structurally relative to the antitumor antibiotic rebeccamycin. Their cytotoxicities toward a number of human tumor cell lines were studied in vitro, and the carbazole N-glycosides were shown to be more active than the bis(indole) glycosides. At the same time, the ribofuranosides were found to be less active than the corresponding ribopyranosides synthesized previously.